The flawless execution of cell division is essential to the generation and survival of all organisms. During every cell cycle, chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of many tumors and birth defects. Chromosomes segregate using their kinetochores, the specialized protein structures that assemble on centromeric DNA sequences and mediate attachment to microtubules. The foundation of all eukaryotic kinetochores is a conserved, inner centromere structure characterized by centromeric chromatin and its associated proteins. A hallmark of centromeric chromatin is Cenp-A, an essential histone H3 variant that epigenetically marks centromeres and is required for kinetochore assembly. The surrounding pericentromeric chromatin also makes various contributions to the fidelity of segregation. To fully understand the mechanisms that ensure accurate chromosome segregation, it is critical to elucidate outstanding questions about the functions and maintenance of centromeric and pericentromeric chromatin. This proposal will use purified kinetochores and a combination of biochemical, biophysical, proteomic and genomic approaches to address a number of outstanding questions about the contribution of centromeric chromatin to kinetochore function. 1) How do centromere-binding proteins contribute to the diverse functions of kinetochores? 2) How does pericentromeric chromatin regulate chromosome segregation? 3) What are the mechanisms that contribute to the exclusive localization of centromeres? The proposal will use budding yeast for these studies because they are amenable to biochemical, genetic and cytological studies, and the yeast kinetochore is the best characterized to date. Taken together, these studies of kinetochores and the underlying chromatin foundation in budding yeast will lead toward an understanding of the fundamental mechanisms of segregation in all eukaryotes. This work will not only elucidate important aspects about the process of segregation, but will aid in the design of better therapeutic interventions in the long-term.